Molluscs of the genus Conus produce a highly toxic venom which enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom which is injected by means of a highly specialized venom apparatus, described by Kohn, A. J., et al, Ann.N.Y. Acad.Sci., 90, 706 (1960). The venom is injected into the prey by means of a disposable hollow tooth which functions both in the manner of a harpoon and a hypodermic needle. The most venomous of all Conus species is Conus geographus L., a fish-hunting species from the central Indo-Pacific. Its venom is so highly toxic that human fatalities have resulted from its sting. Another species, Conus magus, produces a conotoxin having a similar biological activity and structure.
Initial studies on the nature of C. geographus toxicity indicated that death was the result of acute muscle paralysis. Endean, R., et al. Toxicon, 14, 267 (1976) reported that C. geographus toxin inhibited the muscle action potential. Subsequently, attempts were made to fractionate and to partially purify active material from the crude venom (Spence, I., et al, Life Sciences, 21, 1759 (1977); Cruz, L. J., et al, Arch.Biochem.Biophys., 190, 539 (1978)). Such studies revealed that C. geographus venom contains several toxic substances. These substances were shown to be peptides, although their structure was not determined. The toxic peptides of crude venom exhibit a variety of toxic effects. At least one has been found which inhibits muscle action potential. Others were found to act at the neuromuscular junction to inhibit the passage of an excitatory impulse across the junction, but which had no effect on muscle action potential. Conotoxin GI was mischaracterized in the prior art as a direct inhibitor of muscle contraction (Endean, et al, supra, and Cruz, et al, supra), because of the presence of contaminating substances in the toxin preparations.
The present invention discloses for the first time the biological activity and chemical structure of three homologous toxic peptides and their synthesis. The peptides are useful for the treatment of neuromuscular disorders and for rapid reversible immobilization of muscles in all vertebrate species, including humans, thereby facilitating the setting of fractures and dislocations. The toxins inhibit synaptic transmission at the neuromuscular junction and bond strongly to the acetylcholine receptor of the muscle end plate. The toxins are therefore especially suitable for detection and assay of acetylcholine receptors. Such measurements are of significance in clinical diagnosis of myasthenia gravis (Fambrough et al, Science 182 pp 293-295 (1973)). Toxin synthesized with radioactive label, or as a fluorescent derivative provides improved quantitation and sensitivity of acetylcholine receptor assays. All abbreviations used herein not otherwise identified are standard abbreviations approved for publication in the Journal of Biological Chemistry (all amino acids are in the L-configuration unless specifically stated otherwise).